An Integrated Cardiac Microtissue Proteome Map Extends Therapeutic Remodeling by Nanovesicles

Human cardiac microtissues are a promising model to study cardiac biology and disease, but their application is constrained by therapeutic remodeling strategies and limited knowledge of their functional protein expression profiles. Here, we define the use of human cardiac microtissue (hCMT) model generated by assembling induced pluripotent stem cell-derived endothelial cells, cardiac fibroblasts, and cardiomyocytes (CMs) to model ischemia-reperfusion injury (IRI) through a model of hypoxia and reoxygenation and nanovesicle (NV)-mediated functional remodeling. Engineered NVs, generated directly from human stem cells, have been shown to influence cardiac tissue and cell repair, and provide a platform for scalable and reproducible cell free-mediated therapy. We show the functional regulation of the hCMT model and define that administration of NVs (from human induced pluripotent stem cell origin) during reoxygenation significantly increase CM survival and preserve contractility function (contractile duration, relaxation time, and relaxation:contraction velocity). We establish NV uptake and transfer with target cells from the hCMT model. Quantitative proteomics was applied to decipher the cell proteome dynamics and molecular mechanisms of IRI in our in vitro model following NV treatment, linked with networks associated with cell survival, energy production, and stress response regulation. Notably, cell type-specific enrichment analysis revealed that NVs drive distinct proteomic remodeling based on their cell origin, where CERA NVs selectively upregulate cytoprotective and structural networks (such as HSP70, MYH6, and XIRP1) within parenchymal CMs, whereas CL2 NVs predominantly suppress non-myocyte activation and extracellular matrix remodeling factors within the endothelial and fibroblast compartments. Our findings provide an advanced human stem cell-based platform to understand underlying mechanisms of IRI and assess cell-free therapeutic cardioprotective strategies.